Protein phosphatase 2A holoenzyme assembly: Identification of contacts between B-family regulatory and scaffolding a subunits

Abstract

Protein serine/threonine phosphatase (PP) 2A is a ubiquitous enzyme with pleiotropic functions. Trimeric PP2A consists of a structural A subunit, a catalytic C subunit, and a variable regulatory subunit. Variable subunits (B, B′, and B″ families) dictate PP2A substrate specificity and subcellular localization. B-family subunits contain seven WD repeats predicted to fold into a β-propeller structure. We carried out mutagenesis of Bγ to identify domains important for association with A and C subunits in vivo. Several internal deletions in Bγ abolished coimmunoprecipitation of A and C subunits expressed in COS-M6 cells. In contrast, small N- and C-terminal Bγ deletions had no effect on incorporation into the PP2A heterotrimer. Thus, holoenzyme association of B-family subunits requires multiple, precisely aligned contacts within a core β-propeller domain. Charge-reversal mutagenesis of Bγ identified a cluster of conserved critical residues in Bγ WD repeats 3 and 4. Acidic substitution of paired basic residues in Bγ (RR165EE) abolished association with wild-type A and C subunits, while fostering incorporation of Bγ into a PP2A heterotrimer containing an A subunit with an opposite charge-reversal mutation (EE100RR). Thus, binding of A and B subunits requires electrostatic interactions between conserved pairs of glutamates and arginines. By expressing complementary charge-reversal mutants in neuronal PC6-3 cells, we further show that holoenzyme incorporation protects Bγ from rapid degradation by the ubiquitin/proteasome pathway.